Biomolecular Imaging Research Articles

Development of Bright and Biocompatible Nanoruby and Its Application to Background-Free Time-Gated Imaging of G-Protein-Coupled Receptors.

At the forefront of developing fluorescent probes for biological imaging applications are enhancements aimed at increasing their brightness, contrast, and photostability, especially toward demanding applications of single-molecule detection. In comparison with existing probes, nanorubies exhibit unlimited photostability and a long emission lifetime (∼4 ms), which enable continuous imaging at single-particle sensitivity in highly scattering and fluorescent biological specimens. However, their wide application as fluorescence probes has so far been hindered by the absence of facile methods for scaled-up high-volume production and molecularly specific targeting. The present work encompasses the large-scale production of colloidally stable nanoruby particles, the demonstration of their biofunctionality and negligible cytotoxicity, as well as the validation of its use for targeted biomolecular imaging. In addition, optical characteristics of nanorubies are found to be comparable or superior to those of state-of-the-art quantum dots. Protocols of reproducible and robust coupling of functional proteins to the nanoruby surface are also presented. As an example, NeutrAvidin-coupled nanoruby show excellent affinity and specificity to μ-opioid receptors in fixed and live cells, allowing wide-field imaging of G-protein coupled receptors with single-particle sensitivity.

Hydroxyl-Group-Dominated Graphite Dots Reshape Laser Desorption/Ionization Mass Spectrometry for Small Biomolecular Analysis and Imaging.

Small molecules play critical roles in life science, yet their facile detection and imaging in physiological or pathological settings remain a challenge. Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is a powerful tool for molecular analysis. However, conventional organic matrices (CHCA, DHB, etc.) used in assisting analyte ionization suffer from intensive background noise in the mass region below m/z 700, which hinders MALDI MS applications for small-molecule detection. Here, we report that a hydroxyl-group-dominated graphite dot (GD) matrix overcomes limitations of conventional matrices and allows MALDI MS to be used in fast and high-throughput analysis of small biomolecules. GDs exhibit extremely low background noise and ultrahigh sensitivity (with limit of detection <1 fmol) in MALDI MS. This approach allows identification of complex oligosaccharides, detection of low-molecular-weight components in traditional Chinese herbs, and facile analysis of puerarin and its metabolites in serum without purification. Moreover, we show that the GDs provide an effective matrix for the direct imaging or spatiotemporal mapping of small molecules and their metabolites (m/z < 700) simultaneously at the suborgan tissue level. Density functional theory calculations further provide the mechanistic basis of GDs as an effective MALDI matrix in both the positive-ion and negative-ion modes. Collectively, our work uncovered a useful matrix which reshapes MALDI MS technology for a wide range of applications in biology and medicine.

Molecular-dynamics approach for studying the nonequilibrium behavior of x-ray-heated solid-density matter.

When matter is exposed to a high-intensity x-ray free-electron-laser pulse, the x rays excite inner-shell electrons leading to the ionization of the electrons through various atomic processes and creating high-energy-density plasma, i.e., warm or hot dense matter. The resulting system consists of atoms in various electronic configurations, thermalizing on subpicosecond to picosecond timescales after photoexcitation. We present a simulation study of x-ray-heated solid-density matter. For this we use XMDYN, a Monte Carlo molecular-dynamics-based code with periodic boundary conditions, which allows one to investigate nonequilibrium dynamics. XMDYN is capable of treating systems containing light and heavy atomic species with full electronic configuration space and three-dimensional spatial inhomogeneity. For the validation of our approach we compare for a model system the electron temperatures and the ion charge-state distribution from XMDYN to results for the thermalized system based on the average-atom model implemented in XATOM, an ab initio x-ray atomic physics toolkit extended to include a plasma environment. Further, we also compare the average charge evolution of diamond with the predictions of a Boltzmann continuum approach. We demonstrate that XMDYN results are in good quantitative agreement with the above-mentioned approaches, suggesting that the current implementation of XMDYN is a viable approach to simulate the dynamics of x-ray-driven nonequilibrium dynamics in solids. To illustrate the potential of XMDYN for treating complex systems, we present calculations on the triiodo benzene derivative 5-amino-2,4,6-triiodoisophthalic acid (I3C), a compound of relevance of biomolecular imaging, consisting of heavy and light atomic species.

Society of Biomolecular Imaging and Informatics 4th Annual Meeting September 13-15, 2017.

Society of Biomolecular Imaging and Informatics 4th Annual Meeting September 13-15, 2017.

Electronic and Steric Optimization of Fluorogenic Probes for Biomolecular Imaging.

Fluorogenic probes are invaluable tools for spatiotemporal investigations within live cells. In common fluorogenic probes, the intrinsic fluorescence of a small-molecule fluorophore is masked by esterification until entry into a cell, where endogenous esterases catalyze the hydrolysis of the masking groups, generating fluorescence. The susceptibility of masking groups to spontaneous hydrolysis is a major limitation of these probes. Previous attempts to address this problem have incorporated auto-immolative linkers at the cost of atom economy and synthetic adversity. Here, we report on a linker-free strategy that employs adventitious electronic and steric interactions in easy-to-synthesize probes. We find that X···C═O n→π* interactions and acyl group size are optimized in 2',7'-dichlorofluorescein diisobutyrate. This probe is relatively stable to spontaneous hydrolysis but is a highly reactive substrate for esterases both in vitro and in cellulo, yielding a bright, photostable fluorophore with utility in biomolecular imaging.

Review of Proteinochromism Modeling in Computational Chemistry

Understanding and modeling proteinochromism, also known as spectral tuning or color tuning in proteins, receives more and more attention because of the increasing interest in biomolecular imaging, optogenetics and related photochemical tools. The methods of computational chemistry are indispensable tools to support research on proteinochromism but also require the combination of many different techniques, making it a formidable task even for experienced researchers. The field has seen some major improvements recently, especially by an advanced modeling of the protein environment as a polarizable embedding. Here, all relevant aspects of computational proteinochromism modeling, from the basic theory to specific computational considerations, are reviewed and exemplified based on a detailed discussion of theoretical studies on electronic excitations in the green fluorescent protein. Keywords: Fluorescent proteins, theoretical spectroscopy, protein spectroscopy, QM/MM, protein modeling, computational chemistry.

Water in Biomolecular Fluorescence Spectroscopy and Imaging: Side Effects and Remedies.

Historically, many of the classical organic fluorescent dyes were developed as laser dyes and characterized and optimized in organic solvents. Since then, fluorescence has, however, found a vast range of applications in the life sciences in which the fluorophores are usually surrounded by water and not by organic solvents. The omnipresence of water in biomolecular fluorescence spectroscopy and imaging leads to some unwanted but nonetheless unavoidable consequences on the photophysical properties of the dyes, which may impact the quality and complicate quantitative interpretation of the experiments. This paper discusses and illustrates with examples two such water-induced phenomena, namely chromophore aggregation in water and fluorescence quenching by water, as well as some ways to overcome them.

Toward High-Throughput and Multiplexed Imaging of Genome Organization.

Dr. Eric Joyce from the Department of Genetics at the University of Pennsylvania was awarded The President's Innovation award at the annual Society of Biomolecular Imaging and Informatics meeting held in Boston, September 2016. Chromosome interactions are a fundamental aspect of nuclear organization that can activate and silence genes or even direct chromosome rearrangements. However, the molecular mechanisms underlying how chromosomal segments find each other and form stable interactions within cells remain unknown. To address this gap, we have recently developed two technologies that use fluorescent in situ hybridization (FISH) to interrogate chromosome positioning at single-cell resolution. The first is a technology for high-throughput FISH, and the other, called Oligopaints, is a new type of probe that reduces the cost and increases the resolution of FISH. Here, I review our use of these two technologies to uncover and characterize the molecular mechanisms that govern chromosome pairing in Drosophila. I further describe how these methods should benefit a broad spectrum of research fields, including those focusing on chromatin looping, compaction, replication, homologous recombination, and DNA repair.

Assessing the Potential of Metal-Assisted Imaging Mass Spectrometry in Cancer Research.

In the last decade, imaging mass spectrometry (IMS) has been the primary tool for biomolecular imaging. While it is possible to map a wide range of biomolecules using matrix-assisted laser desorption/ionization IMS ranging from high-molecular-weight proteins to small metabolites, more often than not only the most abundant easily ionisable species are detected. To better understand complex diseases such as cancer more specific and sensitive methods need to be developed to enable the detection of lower abundance molecules but also molecules that have yet to be imaged by IMS. In recent years, a big shift has occurred in the imaging community from developing wide reaching methods to developing targeted ones which increases sensitivity through the use of more specific sample preparations. This has been primarily marked by the advent of solvent-free matrix deposition methods for polar lipids, chemical derivatization for hormones and metabolites, and the use of alternative ionization agents for neutral lipids. In this chapter, we discuss two of the latest sample preparations which exploit the use of alternative ionization agents to enable the detection of certain classes of neutral lipids along with free fatty acids by high-sensitivity IMS as demonstrated within our lab.

Multiplexed Single-Cell Imaging: Past, Present, and Future.

Jia-Ren Lin from the Laboratory of Systems Pharmacology at Harvard Medical School was awarded best poster at the annual Society of Biomolecular Imaging and Informatics meeting held in Boston, September 2016. His work focuses on single-cell imaging, especially on developing new methods for simultaneously detecting many antigens, named cyclic immunofluorescence (CycIF). This method could be applied in different stages of drug development, from discovery phase, preclinical research to clinical research. The current works and future directions of CycIF method are summarized in the following overview.

In Situ Electron Microscopy Imaging and Quantitative Structural Modulation of Nanoparticle Superlattices

We use liquid-phase transmission electron microscopy (LP-TEM) to characterize the structure and dynamics of a solution-phase superlattice assembled from gold nanoprisms at the single particle level. The lamellar structure of the superlattice, determined by a balance of interprism interactions, is maintained and resolved under low-dose imaging conditions typically reserved for biomolecular imaging. In this dose range, we capture dynamic structural changes in the superlattice in real time, where contraction and smaller steady-state lattice constants are observed at higher electron dose rates. Quantitative analysis of the contraction mechanism based on a combination of direct LP-TEM imaging, ensemble small-angle X-ray scattering, and theoretical modeling allows us to elucidate: (1) the superlattice contraction in LP-TEM results from the screening of electrostatic repulsion due to as much as a 6-fold increase in the effective ionic strength in the solution upon electron beam illumination; and (2) the lattice constant serves as a means to understand the mechanism of the in situ interaction modulation and precisely calibrate electron dose rates with the effective ionic strength of the system. These results demonstrate that low-dose LP-TEM is a powerful tool for obtaining structural and kinetic properties of nanoassemblies in liquid conditions that closely resemble real experiments. We anticipate that this technique will be especially advantageous for those structures with heterogeneity or disorder that cannot be easily probed by ensemble methods and will provide important insight that will aid in the rational design of sophisticated reconfigurable nanomaterials.

The Society of Biomolecular Imaging and Informatics 3rd Annual Conference High Content 2016 September 12-14, 2016 Joseph B. Martin Conference Center Harvard Medical School, Boston, MA.

The Society of Biomolecular Imaging and Informatics 3rd Annual Conference High Content 2016 September 12-14, 2016 Joseph B. Martin Conference Center Harvard Medical School, Boston, MA.

SBI(2) High Content 2016, 3(rd) Annual Conference, September 12(th)-14(th).

ASSAY and Drug Development TechnologiesVol. 14, No. 7 Society for Biomolecular Imaging and Informatics Third Annual ConferenceSBI2 High Content 2016, 3rd Annual Conference, September 12th-14thJoseph B. Martin Conference Center Harvard Medical School, Boston, MAPublished Online:1 Sep 2016https://doi.org/10.1089/adt.2016.29044.programAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 14Issue 7Sep 2016 InformationCopyright 2016, Mary Ann Liebert, Inc.To cite this article:SBI2 High Content 2016, 3rd Annual Conference, September 12th-14th.ASSAY and Drug Development Technologies.Sep 2016.429-435.http://doi.org/10.1089/adt.2016.29044.programPublished in Volume: 14 Issue 7: September 1, 2016PDF download

Techniques for Accurate Sizing of Gold Nanoparticles Using Dynamic Light Scattering with Particular Application to Chemical and Biological Sensing Based on Aggregate Formation.

Gold nanoparticles (AuNPs) have found broad applications in chemical and biological sensing, catalysis, biomolecular imaging, in vitro diagnostics, cancer therapy, and many other areas. Dynamic light scattering (DLS) is an analytical tool used routinely for nanoparticle size measurement and analysis. Due to its relatively low cost and ease of operation in comparison to other more sophisticated techniques, DLS is the primary choice of instrumentation for analyzing the size and size distribution of nanoparticle suspensions. However, many DLS users are unfamiliar with the principles behind the DLS measurement and are unware of some of the intrinsic limitations as well as the unique capabilities of this technique. The lack of sufficient understanding of DLS often leads to inappropriate experimental design and misinterpretation of the data. In this study, we performed DLS analyses on a series of citrate-stabilized AuNPs with diameters ranging from 10 to 100 nm. Our study shows that the measured hydrodynamic diameters of the AuNPs can vary significantly with concentration and incident laser power. The scattered light intensity of the AuNPs has a nearly sixth order power law increase with diameter, and the enormous scattered light intensity of AuNPs with diameters around or exceeding 80 nm causes a substantial multiple scattering effect in conventional DLS instruments. The effect leads to significant errors in the reported average hydrodynamic diameter of the AuNPs when the measurements are analyzed in the conventional way, without accounting for the multiple scattering. We present here some useful methods to obtain the accurate hydrodynamic size of the AuNPs using DLS. We also demonstrate and explain an extremely powerful aspect of DLS-its exceptional sensitivity in detecting gold nanoparticle aggregate formation, and the use of this unique capability for chemical and biological sensing applications.

Tissue Plasminogen Activator Binding to Superparamagnetic Iron Oxide Nanoparticle-Covalent Versus Adsorptive Approach.

Functionalized superparamagnetic iron oxide nanoparticles are frequently used to develop vehicles for drug delivery, hyperthermia, and photodynamic therapy and as tools used for magnetic separation and purification of proteins or for biomolecular imaging. Depending on the application, there are various possible covalent and non-covalent approaches for the functionalization of particles, each of them shows different advantages and disadvantages for drug release and activity at the desired location.Particularly important for the production of adsorptive and covalent bound drugs to nanoparticles is the pureness of the involved formulation. Especially the covalent binding strategy demands defined chemistry of the drug, which is stabilized by excess free amino acids which could reduce reaction efficiency. In this study, we therefore used tangential flow filtration (TFF) method to purify the drugs before the reaction and used the frequently applied and clinically available recombinant tissue plasminogen activator (tPA; Actilyse®) as a proof of concept. We then coupled the tPA preparation to polyacrylic acid-co-maleic acid (PAM)-coated superparamagnetic iron oxide nanoparticles (SPIONs) using an amino-reactive activated ester reaction and compared these particles to PAM-coated SPIONs with electrostatically adsorbed tPA.Using dynamic light scattering (DLS) and pH-dependent electrokinetic mobility measurements, we showed that surface properties of the SPIONs were significantly greater affected after activation of the particles compared to the adsorption controls. Different in vitro assays were used to investigate the activity of tPA after coupling to the particles and purification of the ferrofluid. Covalent linkage significantly improves the reactivity and long-term stability of the conjugated SPION-tPA system compared to simple adsorption. In conclusion, we have shown an effective way to produce SPIONs with covalent and non-covalent ultra-filtrated drugs. We showed that using activated ester reaction, immobilization of the protein was significantly better than in adsorptive approaches. Investigation of those functionalized SPIONs revealed diverging attributes, which should be taken into account when developing nanoparticles for different applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s11671-016-1521-7) contains supplementary material, which is available to authorized users.

ChemInform Abstract: Raman and Infra‐Red Microspectroscopy: Towards Quantitative Evaluation for Clinical Research by Ratiometric Analysis

AbstractReview: 123 refs.

Polarization-resolved hyperspectral stimulated Raman scattering microscopy for label-free biomolecular imaging of the tooth

We report the development and implementation of a rapid polarization-resolved hyperspectral stimulated Raman scattering (SRS) microscopy technique for label-free biomolecular imaging of the tooth. The hyperspectral SRS imaging technique developed covers both fingerprint (800–1800 cm−1) and high-wavenumber (2800–3600 cm−1) regions for tooth Raman imaging without fluorescence background interference with an imaging speed of &amp;lt;0.3 s per frame of 512 × 512 pixels (∼1 μs per pixel), that is, &amp;gt;106 faster than confocal Raman imaging. Significant differences of hyperspectral SRS spectra among different tooth locations (e.g., dentin, enamel, and dentin-enamel junction) are observed, revealing the biochemical distribution differences across the tooth. Further polarization-resolved SRS imaging shows different polarization dependences related to the molecular orientation differences of various tooth locations. This work demonstrates the potential of polarization-resolved hyperspectral SRS imaging technique developed in rapidly characterizing biochemical structures and compositions as well as biomolecule organizations/orientations of the tooth without labeling.

Hyperlensing at NIR frequencies using a hemispherical metallic nanowire lens in a sea-urchin geometry.

Label-free and real time far-field super-resolution imaging (hyperlensing) at the nanoscale is of significant interest where sub-λ imaging remains a constraint because of Abbe's diffraction limit. Though by utilizing anisotropic permittivities, metal-dielectric multilayers have been successful in reconstructing high-frequency components of sub-λ objects, yet they remain cumbersome and expensive to make. Most of the multilayer structures require multiple vacuum deposition cycles and are plagued by stringent requirements on the surface roughness of metallic layers. In contrast to the multilayer structure here we propose a 3D hyperbolic metamaterial model composed of metallic nanorods arranged in a sea-urchin geometry as a hyper-lensing device, which is capable of projecting and magnifying diffraction limited information into the far-field at Near-infrared (NIR) frequencies. The hyperlens generates a band of flat hyperbolic dispersions in spherical coordinates, which in turn supports the propagation of high wave-vector spatial harmonics leading to far-field super-resolution imaging. Using full-wave finite-difference time-domain (FDTD) simulations with diffraction limited trimer, quadrumer and ringed objects etched on thin perfect electric conductor (PEC) films, we show that the hyperlens model can achieve magnification factors of up to 10× in the far-field (∼4.5λ from the object's surface) under a light source with a wavelength of 1 μm, with successful resolution down to 220 nm (∼λ/5). The magnified image field distribution projected into the far-field is shown to follow the object under a reduction in the symmetry. These results are important for making progress in the realization of real-time biomolecular imaging systems, eliminating the need for near-field scanning, destructive electron microscopy and various image post-processing techniques.

Ultrastructural Mapping of the Zebrafish Gastrointestinal System as a Basis for Experimental Drug Studies.

Research in the field of gastroenterology is increasingly focused on the use of alternative nonrodent model organisms to provide new experimental tools to study chronic diseases. The zebrafish is a particularly valuable experimental platform to explore organ and cell structure-function relationships under relevant biological and pathobiological settings. This is due to its optical transparency and its close-to-human genetic makeup. To-date, the structure-function properties of the GIS of the zebrafish are relatively unexplored and limited to histology and fluorescent microscopy. Occasionally those studies include EM of a given subcellular process but lack the required full histological picture. In this work, we employed a novel combined biomolecular imaging approach in order to cross-correlate 3D ultrastructure over different length scales (optical-, X-ray micro-CT, and high-resolution EM). Our correlated imaging studies and subsequent data modelling provide to our knowledge the first detailed 3D picture of the zebrafish larvae GIS. Our results provide unequivocally a limit of confidence for studying various digestive disorders and drug delivery pathways in the zebrafish.

Lipoprotein Nanoplatelets: Brightly Fluorescent, Zwitterionic Probes with Rapid Cellular Entry.

Semiconductor nanoplatelets (NPLs) are planar nanocrystals that have recently attracted considerable attention due to their quantum-well-like physics, atomically precise thickness, and unique photophysical properties such as narrow-band fluorescence emission. These attributes are of potential interest for applications in biomolecular and cellular imaging, but it has been challenging to colloidally stabilize these nanocrystals in biological media due to their large dimensions and tendency to aggregate. Here we introduce a new colloidal material that is a hybrid between a NPL and an organic nanodisc composed of phospholipids and lipoproteins. The phospholipids adsorb to flat surfaces on the NPL, and lipoproteins bind to sharp edges to enable monodisperse NPL encapsulation with long-term stability in biological buffers and high-salt solutions. The lipoprotein NPLs (L-NPLs) are highly fluorescent, with brightness comparable to that of wavelength-matched quantum dots at both the ensemble and single-molecule levels. They also exhibit a unique feature of rapid internalization into living cells, after which they retain their fluorescence. These unique properties suggest that L-NPLs are particularly well suited for applications in live-cell single-molecule imaging and multiplexed cellular labeling.